The goal of this revised R01 application is to further optimize the DNA prime and protein boost approach for the development of improved polyvalent HIV vaccine formulations that can generate broad neutralizing antibodies against targeted HIV-1 viruses. This application is based on our recent results that the DNA prime plus protein boost immunization approach was effective in eliciting neutralizing antibody responses against certain primary HIV isolates across multiple clades from both preclinical animal and early phase human clinical studies. This was achieved when the Env antigens from primary HIV-1 isolates were used in this prime/boost vaccination approach. It is believed that DNA priming contributes to the quality of the final antibody responses while the protein boost leads to a quick production of high level antibody responses. Because DNA immunization has been traditionally used for cell mediated immune responses rather than antibody responses, this unexpected finding will bring novel applications to DNA immunization in eliciting neutralizing antibodies against HIV-1. Therefore our approach has exciting potential in human HIV vaccine development. Aim 1. To further expand the breadth of neutralizing antibodies against additional primary viral isolates especially those resistant to our pilot polyvalent formulations. Aim 2. To develop the next generation DNA prime and protein boost HIV vaccines by using optimized forms of Env antigen designs and optimized immunization schedules. Aim 3. To study the immunological mechanism and the antibody affinity maturation process that may contribute to the efficacy of DNA prime plus protein boost strategy so that more effective polyvalent DNA plus protein vaccines can be developed. The current application combines two important technologies in HIV vaccine development: the DNA prime plus protein boost vaccination approach which has been shown promise in generating neutralizing antibodies against primary HIV-1 isolates and the high throughput neutralization assays with standardized primary viral panels. This will allows us to effectively screen a large number of unique primary Env antigens to formulate the effective polyvalent HIV vaccines for the next phase of clinical testing.